The invention relates to novel conjugates of tetrasaccharides, preferably of sialyl-Lewis X (SLeX) and sialyl-Lewis A (SLeA), with improved action as inhibitors of cell adhesion, a process for the preparation of these compounds, and their use as pharmacological active compounds and as diagnostics, and pharmaceuticals which contain these conjugates.
The circulation of blood cells, e.g. leucocytes, neutrophils, granulocytes and monocytes is, on a molecular plane, a multistage, very complex process which is only known in individual steps (Review: T. A. Springer, Cell 76, 301-314, 1994).
The most recent research results showed that the recirculation of the lymphocytes crucial in immune monitoring and the localization of neutrophils and monocytes at inflammatory foci respond to very similar molecular mechanisms. Thus in acute and chronic inflammatory processes adhesion of the leucocytes to endothelial cells and migration into the inflammatory focus and into the secondary lymphatic organs occurs.
This process involves numerous specific signal molecules, e.g. interleukins, leucotrienes and tumor necrosis factor (TNF), receptors coupled to their G protein and in particular tissue-specific cell adhesion molecules, which guarantee a specifically controlled recognition of the immune and endothelial cells. The most important adhesion molecules involved in this process, which in the following will be designated as receptors, include the selectins (E-, P- and L-selectins), integrins and the members of the immunoglobulin superfamily.
The three selectin receptors determine the starting phase of leucocyte adhesion. E-selectin is expressed on endothelial cells a few hours after stimulation, for example by interleukin-1 (IL-1) or tumor necrosis factor .alpha. (TNF-.alpha.), while P-selectin is stored in blood platelets and endothelial cells and is presented on the cell surfaces after stimulation by thrombin, peroxide radicals or substance P among others. L-selectin is continuously expressed on leucocytes.
It is today generally recognized that the tetrasaccharides sialyl-Lewis X (SLeX) and sialyl-Lewis A (SLeA) which occur on cell membranes as substructures of glycosphingolipids and glycoproteins, can function as ligands for all three selectin receptors. (Review: A. Giannis, Angew. Chem. 106, 188, 1994): ##STR1## The regioisomeric compound sialyl-Lewis A is closely related to the X type and binds to selectin receptors with comparable affinity. The A type arises from the X type by simple exchange of the "side chains" on the central N-acetylglucosamine unit: ##STR2##
The course of a number of acute and chronic disorders is unfavorably affected by the excessive adhesion of leucocytes and their infiltration into the tissue affected. These include, for example, rheumatism, reperfusion injuries such as myocardial ischemia/infarct (MI), acute pneumonia after operative intervention, traumatic shock and stroke, psoriasis, dermatitis, ARDS (adult respiratory distress syndrome) and the restenosis occurring after surgical intervention (for example angioplasty).
A very promising therapeutic starting point is therefore the attempt to employ the tetrasaccharides SLeX/A in various administration forms or mimetics thereof having a modified structure as antagonists for the modulation or suppression of excessive leucocyte adhesion and to employ them for the alleviation or cure of said disorders.
The natural ligand having the structure of SLeX has already been successfully used in animal experiments in P-selectin-dependent lung injuries (M. S. Mulligan et al., Nature 1993, 364, 149) and in myocardial reperfusion injuries (M. Buerke et al., J. Clin. Invest. 1994, 93, 1140). In primary clinical trials in acute pneumonia the compound should be employed in a dose of 1 to 2 grams per day per patient (communication of Cytel Corp./La Jolla (CA.) in the 2nd Glycotechnology Meeting/CHI in La Jolla/USA on May 16-18th 1994). This high dose of active compound is in agreement with the, as is known, weak affinity of the natural SLeX/A ligands for the selectin receptors. Thus SLeX in all known in vitro test systems inhibits cell adhesion to selectin receptors only at a relatively high concentration in the range of IC.sub.50 =1 to 3 mM.
In some publications, meanwhile, efforts to obtain more strongly binding antagonists by structural variation of the ligand have been reported. The variation of the fucose and neuraminic acid units until now regarded as crucial for the structure-activity relationship (B. K. Brandley et al., Glycobiology 1993, 3, 633 and M. Yoshida et al., Glycoconjugate J. 1993, 10, 3), however, did not afford any significantly improved inhibition values. Only on variation of the glucosamine unit (replacement of GlcNAc by glucose and azido and amino groups in the 2-position of GlcNAc) could a significantly increased affinity to the E-selectin receptor be achieved. The IC.sub.50 data of these compounds which can be prepared by complete de novo synthesis should be 0.12 mM (compared with 1.2 to 2.0 mM for SLeX) for the inhibition of the adhesion of HL-60 and U-937 cells with E-selectin. A disadvantage, however, is that the binding to L- and P-selectins at &gt;5 mM is severely impaired (Dasgupta et al., poster presentation of Glycomed Inc. on the occasion of the conference in La Jolla in 5/94).
In another in vitro test system, in which, with reversal of the natural physiological conditions, the soluble receptor construct E-selectin-IgG (instead of the corresponding receptor construct in immobilized form, which would more likely be comparable with the natural situation on endothelial cells) binds to the immobilized ligands and is displaced by potential inhibitors, in the system E-selectin-IgG/immobilized BSA-SLeA a 36-fold higher affinity was found for the ligand with N-acetyl-glucosamine deacetylated in the 2-position.
Apart from a restricted comparability of this artificial test system with the situation in vivo, i.e. the inhibition of the adhesion of cells which express the natural ligands SLeX/A, this result remains restricted to the E-selectin receptor, for with the P-selectin receptor only weak inhibition effects were found at inhibitor concentrations of about 1 mM (R. M. Nelson et al., J. Clin. Invest. 1993, 91, 1157).
The prior art on the binding affinity of modified SLeX/A structures to selectins is referred to in Pharmacochem. Libr. 1993, 20 (Trends in Drug Research), pages 33-40.
Modified ligands of the SLeX/A structural type, which are mainly derived from the lactose and from the lactosamine basic structure and could be employed, inter alia, as potential selectin antagonists, are claimed in several patent applications, in particular in the international publications
WO 91/19501, WO 91/19502, WO 92/02527, WO 93/10796, PA0 WO 94/00477, WO 92/18610, WO 92/09293, WO 92/07572, PA0 WO 92/16640, WO 92/19632, WO 93/17033, WO 93/23031, PA0 WO 92/22301, WO 92/22563, WO 92/22564, WO 92/22565, PA0 WO 92/22661, WO 92/22662, WO 93/24505, WO 93/24506.
SLeX/A derivatives or mimetics having clearly improved affinity for the E-selectin and for the P-selectin receptor in vitro have still not been described. What are remarkable, however, are indications that multivalent ligands could have a higher binding affinity compared with monovalent ligands: thus an enzymatically prepared, complex nonasaccharide binds 5 times better (IC.sub.50 =0.4 mM) to E-selectin as a potentially divalent ligand than the monomeric SLeX ligand. On closer analysis, this value, however, does not represent a convincing multivalent effect: if it is considered that the IC.sub.50 value calculated per ligand is actually only 0.8 mM, then no significant improvement was actually achieved (S. A. DeFrees et al., J. Am. Chem. Soc. 1993, 115, 7549).
A further possibility for the multiple presentation of the SLeX/A ligands consists in the introduction of (co)polymerizable side chains or in the binding of a suitable SLeX precursor to a multifunctional polymer. The first variant leads to artificial polymer conjugates, for example to polyacrylamides, which are unsuitable as pharmaceuticals on account of their physiological intolerability. In the literature, this procedure was described for polyacrylamide conjugates of the Lewis X trisaccharide (S.-I. Nishimura et al., Macromolecules 1994, 27, 157). The process can also be applied to SLeX, SLeA and analogs in which the sialic acid has been replaced by sulfate (E. Nifantev, Lecture, Glycotechnology Meeting, La Jolla, May 16th-18th, 1994).
The second variant, in which SLeX derivatives are reacted with a reactive polymer to give multifunctional, biocompatible and physiologically tolerable polymer conjugates, is described in EP 0 601 417. In this publication reference is also made in detail to the prior art on carbohydrate polymer conjugates.
An inherent disadvantage of these carbohydrate polymer conjugates with respect to their utility as pharmaceuticals lies in the polymer nature of active compounds of this type: in each synthetic batch a new type of product is obtained which is characterized by a differing molecular weight distribution and by a variable coating density of the carbohydrate ligand bound to the polymer.
In WO 94/00477, it is proposed to prepare multivalent compounds by reductive amination of the oxime adducts of ligands of the structural type (1), which are present as free oligosaccharides (i.e. R.sup.1 .dbd.OH), with peptides or proteins. This process, however, has serious disadvantages, as the SLeX ligand is severely modified by the ring opening of the first carbohydrate unit at the reducing end (GlcNAc or Glc). Additionally, as in the polymer conjugates mentioned, only inexactly defined and uncharacterizable mixtures are to be expected. This is shown plainly in the example described hypothetically in WO 94/00477, in which using a 10,000-fold excess (1 mmol) of useful oligosaccharide a coupling to the tripeptide Lys-Tyr-Lys on the analytical scale of 0.1 .mu.mol of Lys-Tyr-Lys tripeptide is proposed. After carrying out analytical separation processes, the possibility of an analysis by means of mass spectroscopy is mentioned, according to which mixtures of mono-, di- and trivalent carbohydrate conjugates are expected.
Low molecular weight compounds are therefore desirable having an unequivocal empirical formula and defined molecular weight and having a distinctly increased receptor affinity. Compounds are particularly desirable which simultaneously meet all the requirements mentioned and moreover can be prepared on the preparative scale (gram amounts) in efficient synthetic processes.
The object of the invention is to provide low molecular weight carbohydrate receptor blockers, a simple process for their preparation, and pharmaceuticals prepared from these which meet the requirements mentioned.
This object is achieved according to the invention by the simple coupling of oligosaccharides which can be carried out in a few steps and in high yields, preferably with SLeX/A structures, to mono- and trifunctional precursors to give novel carbohydrate conjugates. The useful oligosaccharide component can be employed stoichiometrically or in a small excess of 1 to 10 mol % per active group and preferably in unprotected form.
Surprisingly, in the synthetic route according to the invention to trivalent carbohydrate conjugates, only small amounts of the divalent by-products are formed, which can be separated off easily from the desired main products.
The finding is additionally surprising that the novel conjugates bind to E- and P-selectins more strongly than the natural ligand of the formula (1). The agonistic and antagonistic action of the compounds according to the invention can be employed for the prophylaxis, therapy and diagnosis of disorders which are characterized by excessive cell-cell adhesion.